In vivo high-resolution structural imaging of large arteries in
small rodents using two-photon laser scanning microscopy
Citation for published version (APA):Megens, R. T. A., Reitsma, S., Prinzen, L., Oude Egbrink, M. G. A., Engels, W., Leenders, P. J. A., Brunenberg, E. J. L., Reesink, K. D., Janssen, B. J. A., Haar Romeny, ter, B. M., Slaaf, D. W., & Zandvoort, van, M. (2010). In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning
microscopy. Journal of Biomedical Optics, 15(1), 011108-1/10. https://doi.org/10.1117/1.3281672
DOI:
10.1117/1.3281672
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In vivo high-resolution structural imaging of large
arteries in small rodents using two-photon laser scanning
microscopy
Remco T. A. Megens*
Sietze Reitsma*
Lenneke Prinzen Maastricht University
Department of Biomedical Engineering Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands Mirjam G. A. oude Egbrink Maastricht University
Department of Physiology
Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands Wim Engels
Maastricht University
Department of Biomedical Engineering Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands Peter J. A. Leenders
Maastricht University Pharmacology and Toxicology
Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands Ellen J. L. Brunenberg
University of Technology
Department of Biomedical Engineering WH 1.105, P.O. Box 513
Eindhoven, 5600 MB, The Netherlands Koen D. Reesink
Maastricht University
Department of Biomedical Engineering Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands Ben J. A. Janssen
Maastricht University Pharmacology and Toxicology
Cardiovascular Research Institute Maastricht Maasticht, 6200 MD, The Netherlands Bart M. ter Haar Romeny University of Technology
Department of Biomedical Engineering WH 1.105, P.O. Box 513
Eindhoven, 5600 MB, The Netherlands
Dick W. Slaaf Maastricht University
Department of Biomedical Engineering Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands
and
University of Technology
Department of Biomedical Engineering WH 1.105, P.O. Box 513
Eindhoven, 5600 MB, The Netherlands Marc A. M. J. van Zandvoort Maastricht University
Department of Biomedical Engineering Cardiovascular Research Institute Maastricht Maastricht, 6200 MD, The Netherlands
Abstract. In vivo共molecular兲 imaging of the vessel wall of
large arteries at subcellular resolution is crucial for unrav-eling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy 共TPLSM兲 in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with car-diac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is per-formed on rat renal and mouse carotid arteries, both sur-gically exposed and labeled fluorescently 共cell nuclei, elastin, and collagen兲. The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. In-deed, structures in the vessel wall 共cell nuclei, elastic laminae兲 can be imaged at subcellular resolution. In me-chanically damaged carotid arteries, even the subendothe-lial collagen sheet共⬃1m兲 is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying共diseased兲 arteries
in vivo or immediate validation of in vivo molecular
im-aging techniques such as magnetic resonance imim-aging 共MRI兲, ultrasound, and positron emission tomography 共PET兲. © 2010 Society of Photo-Optical Instrumentation Engineers.
关DOI: 10.1117/1.3281672兴
Keywords: in vivo imaging; two-photon microscopy; subendothelial collagen; triggering; quantum dots; heart rate; respiration rate.
Paper 09117SSR received Apr. 4, 2009; revised manuscript received Aug. 14, 2009; accepted for publication Sep. 1, 2009; published on-line Jan. 25, 2010.
1 Introduction
In the past decades, imaging of 共patho兲 physiological pro-cesses in large共conduit兲 arteries has become increasingly
im-1083-3668/2010/15共1兲/011108/10/$25.00 © 2010 SPIE *Authors contributed equally to this work.
Address all correspondence to: Marc A.M.J. van Zandvoort, PhD, Department of Biomedical Engineering, Maastricht University, P.O. Box 616, 6200 MD Maas-tricht, The Netherlands; Tel: 31-43-3881200; Fax: 31-43-3884166; E-mail: MAMJ.vanZandvoort@BF.unimaas.nl
portant in the context of diseases such as atherosclerosis. Re-cently, we and others have put much effort into developing techniques to visualize vascular structure and function espe-cially in large murine vessels. We recently published an ex
vivo setup based on two-photon laser scanning microscopy
共TPLSM兲, which enables structural and functional fluorescent imaging of intact, viable larger arteries of mice at a subcellu-lar resolution.1 However, real-time in vivo imaging in these vessels at such a resolution has not been reported yet.
Conventional intravital bright-field and fluorescence mi-croscopy techniques are particularly suitable for imaging small blood vessels in thin transparent tissues where detection of fluorescent blood cells is possible up to a depth of100m. They enable among others advantages, visualization of blood cell-vessel wall interactions2,3and hemodynamic parameters.4 It was demonstrated that conventional intravital fluorescence microscopy also allows for detection of fluorescently labeled blood cells that interact with the vessel wall in large 共athero-sclerotic兲 arteries in mice5–8共diameter up to 600m兲. How-ever, this technique is not suitable for imaging共subcellular兲 structures in the vessel wall of these large arteries. Image quality is strongly hampered by contributions from out-of-focus and scattered fluorescence, and rapidly decreases at deeper layers. This precludes the use of conventional intravi-tal fluorescence microscopy for in vivo visualization of sub-cellular structures in the vessel wall of larger arteries.
Confocal laser scanning microscopy offers excellent high-resolution images of vascular structures in vivo.9,10However, spatial resolution gradually deteriorates deeper in the sample 共⬎40m兲 due to out-of-focus scattered fluorescence light passing through the pinhole. Furthermore, the focal-to-nonfocal signal ratio decreases rapidly with depth, leading to poor penetration depth.11In contrast, the spatial resolution of TPLSM is less affected by scattering. Two-photon excitation of fluorescent molecules uses a wavelength that is twice that of single photon excitation. This higher excitation wavelength is less susceptible to scattering. Furthermore, two-photon ex-citation occurs only in a small volume element共voxel兲 in the focal plane. Any light reaching the objective lens originates from the excited voxel, independent of the amount of scatter-ing experienced by the emitted light. This results in preserva-tion of spatial resolupreserva-tion in scattering tissues and optical sec-tioning. The combination of increased penetration depth, good optical sectioning, and subcellular resolution enables ex vivo TPLSM imaging of large arteries at high spatial resolution.1,11–15 Also in vivo TPLSM has been reported in various relatively motionless tissues and structures, such as brain,16 kidney,17–21 microvasculature,22 skin,23,24 and lymphatics.25
The complication of imaging of large arteries in vivo origi-nates from cardiac and respiratory activity, which results in a repetitive but variable pattern of vessel movement. Random acquisition of images within this pattern will result in images that reflect different parts of vessel motion 共between-frame motion artifacts兲. In this way, such images contain random parts of the vessel and are not mutually comparable. Further-more, part of the vessel movement is captured within each image, resulting in image distortion 共in-frame motion arti-facts兲. In conventional fluorescence microscopy, image distor-tion can be eliminated by using flashed illuminadistor-tion at the
right period during the cardiac and respiratory cycles. Unfor-tunately, TPLSM does not allow instantaneous image acquisi-tion, since image composition is based on point-by-point scanning through the optical section. The diameter of large arteries in small rodents is about 400 to 600m, and their distention is significantly larger than the thickness of the op-tical section, which is only about 1.5m in TPLSM. This induces image distortion and challenges its use in imaging of large arteries in vivo.11 Some researchers have evaded this problem by temporarily stopping blood flow.26This approach, however, severely disrupts the physiological conditions in these vessels. Moreover, it cannot be used to visualize dy-namic processes such as blood cell-vessel wall interactions. In other imaging modalities, such as magnetic resonance imag-ing, triggered image acquisition is used to overcome these motion artifacts.
In this study, we propose and test whether for applications of TPLSM in vivo, motion artifacts can be minimized by mak-ing several adaptations to the acquisition mode. one is whether acceleration of image acquisition will decrease image distortion. This can be achieved by reducing the number of pixels or the pixel dwell-times共or a combination of both兲. A drawback of accelerated image acquisition is a reduction of signal-to-noise ratio 共SNR兲. Second, the acquisition of each separate image in a time sequence should start at a specific, fixed moment of the cardiac and respiration cycle. This poten-tially results in a series of images that all contain a similar part of the moving arterial wall, thus limiting between-frame motion artifacts. Finally, the application of specific image pro-cessing tools may improve overall image quality.
Imaging was performed in surgically exposed arteries of anesthetized mice and rats, which were stained with specific fluorescent markers. As a proof of principle for the method and its applicability for testing molecular imaging agents in
vivo, damage-induced exposure of the thin subendothelial
col-lagen sheet was visualized using colcol-lagen-targeted quantum dots.
2 Materials and Methods
2.1 Animals
All experiments were in line with institutional guidelines and approved by the local ethics committee on the use of labora-tory animals. Mice共C57BL6/J; n=7兲 were anesthetized using subcutaneous administration of a mixture of 75 mg/kg ket-amin 共Nimatek, Eurovet, Cuijck, The Netherlands兲 and 15 mg/kg xylazin 共Xylazin, Ceva Sante Animale BV, Naald-wijk, The Netherlands兲 in a volume of 3 ml/kg. Anesthesia was maintained by subcutaneous injection of0.88 ml/kg of the same xylazin-ketamin mixture every 30 min. Rats 共Wistar-Kyoto; n=6兲 were anesthetized by a single intraperi-toneal injection of urethane共1.7 mg/g兲. All anesthetics were dissolved in saline. Body temperature was monitored using a rectal probe 共PT100 sensor, Watlow, United Kingdom兲 and maintained at approximately 37 ° C by a heating platform 共TH60-SMZ, Linkam Scientific Instruments, United King-dom兲.
2.2 Tissue Preparation and Instrumentation
Animals were placed in the supine position. In mice, the right common carotid artery was surgically exposed up to the bifurcation.5 In rats, the abdominal region was surgically opened to expose the left renal artery. To simplify discrimina-tion between artery and surrounding tissues in both prepara-tions, a thin black plastic sheet was placed underneath the artery segments without stretching the vessel. Exposed areas were kept moist with saline at all time during the experiment. In both species, the left jugular vein was cannulated for administration of fluorescent dyes. Blood pressure was mea-sured through a catheter inserted in the left femoral artery and linked to two blood pressure sensors共Baxter Uniflow, Baxter B.V., Utrecht, The Netherlands兲; the first sensor was used to monitor blood pressure and, hence, the hemodynamic condi-tion of the animal. The second blood pressure signal was used as input for image triggering共see later in the paper兲. All cath-eters and blood pressure probes were filled with heparin 共5 U/ml in saline兲.
To stabilize respiration, the trachea was intubated and ven-tilated with normal air using a ventilator 共mouse: Minivent 845, Hugo Sachs Electronic GmbH, Germany; rat: Harvard rodent respirator, Harvard Apparatus, Massachusetts兲. As heart rate and also vessel motion may vary slightly from changes in intrathoracic pressure due to breathing, a respira-tion sensor共Graseby, Wicklow, Ireland兲 was placed on top of the chest to obtain a共relative兲 respiration signal for the trigger unit. In a number of mice共n=3兲, no 共forced兲 ventilation was used to investigate the effect of spontaneous breathing on im-age motion and triggering.
2.3 Fluorescent Labeling
Nuclei of viable cells were labeled by topical and/or intrave-nous administration of the viable cell membrane permeable DNA/RNA markers SYTO13 or SYTO41共Molecular Probes, Leiden, The Netherlands兲. Eosin 共Molecular Probes兲 was used as a specific fluorescent marker for elastin. SYTO13 and SYTO41共both 2.0M兲 and eosin共0.5M兲 were dissolved in saline and administered topically on the artery. In four rats and three mice, SYTO13 was infused intravenously共0.1 ml of a4.0M solution in saline兲 to more specifically stain the endothelial cell nuclei and blood cells. Circulating blood platelets, cytoplasm of leukocytes, and cells in the arterial wall were labeled fluorescently by intravenous administration 共i.v.兲 of acridin red 共Chroma-Gesellschaft Schmidt GmbH, Germany; 2.0 mg/ml in saline with 5% ethanol; a bolus of 0.03 ml in mice or 0.1 ml in rats兲. Collagen 共types I and III兲 in the tunica adventitia was visualized by second-harmonic generation 共SHG兲. The subendothelial collagen layer 共type IV兲 is too thin and located too deep to be detected by SHG on our system, especially in vivo.1,2 Therefore, subendothelial collagen, which is luminally exposed on damage to the endot-helium, was visualized using collagen-specific CNA3512,27 la-beled with green-fluorescent quantum dots 关CNA35-QD525; 200l i.v. containing 15M CNA35-biotin and 0.5M streptavidin coated quantum dots共Invitrogen兲兴. Propidium io-dide was used to label nonviable cells共PI; Invitrogen; 200l of a0.01 mg/ml solution applied topically兲.
2.4 Damage-Induced Subendothelial Collagen Exposure
In a subset of experiments, the carotid artery of mice was pinched with a fine-tipped forceps for 3 s to induce damage across the vessel wall. In our ex vivo setup, this procedure has been shown to be effective both in inducing cell damage across the vessel wall,1and in uncovering the subendothelial collagen layer.12 Then, the CNA35-QD525 solution was in-fused i.v. to label exposed subendothelial collagen.27 Further-more, propidium iodide was applied topically to label nonvi-able cells.
2.5 Image Acquisition
A Nikon E600FN microscope共Nikon Corporation, Tokyo, Ja-pan兲 connected to a Biorad 2100 MP multiphoton system 共Biorad, Hemel Hempstead, United Kingdom兲 was used for TPLSM as previously described.1,28
A 120⫾20-fs-pulsed Ti:sapphire laser 共Spectra Physics Tsunami, Mountain View, California兲 was used as the excita-tion source tuned and mode-locked at 800 to 840 nm. Exci-tation powers at the sample varied from60 to 80 mW. Either a40⫻ or a 60⫻ water dipping objective was used 关numerical aperture共NA兲 0.8 and 1.0, respectively兴 for TPLSM record-ings. A20⫻ water dipping objective 共NA 0.5兲 was used in combination with a 20-W halogen cold light source 共Schott KL 200, Schott AG, Mainz, Germany兲 to locate the exposed vessels.
To detect the emitted fluorescent signals, three photomul-tiplier tubes were used. These were tuned to correspond with parts of the emission spectra of the fluorescent markers ap-plied, aiming to find a balance between minimal bleed-through versus maximal signal reception.1
Images of256⫻ 256 pixels were obtained for each color channel and combined into one RGB image. Series of 20 to 100 subsequent xy images were recorded共time series兲. Acqui-sition of each separate image was started by a trigger pulse derived from a trigger unit共see later in the paper兲. Optimal image acquisition time was determined empirically depending on heart rate共see later in the paper兲.
Table1summarizes possible image acquisition times and settings of the system. Image acquisition times applied in this paper varied from0.11 to 0.43 s, depending on matrix size, pixel dwell-time, line speed, and artery/species. When imag-ing at⬎500 lines/s, optical zoom is automatically applied to reduce total scan area, and hence, image acquisition time. As an advantage, spatial resolution is increased. Furthermore, line speeds of ⬎750 lines/s are achieved through bidirec-tional scanning, which increases scanning speed共requiring no or less optical zoom兲, and also results in 共correctable兲 inter-lacing errors共see later in the paper兲. Image acquisition time can also be decreased by using double and quadruple line scanning modes, where two or four lines are scanned at the same time. This results in a decrease of spatial resolution in the vertical direction共y direction兲, which can be corrected by applying additional zoom at the cost of FOV. Objects of in-terest should therefore be aligned in such a way that the maxi-mum spatial resolution is obtained in the required direction.
Table1 Image acquisition characteristics. Image Acquisition Time共s兲
Scanning mode
Line Scan Rate
共lines s−1兲 Optical Zoom
Maximum FOV 共m2兲 1⫻ 2⫻ 4⫻ 10.24 5.12 2.56 25 1.0 206⫻206 5.12 2.56 1.28 50 1.0 206⫻206 1.54 0.77 0.39 166 1.0 206⫻206 0.51 0.26 0.13 500 1.0 206⫻206 0.43 0.21 0.11 600 1.2 172⫻172 0.34 0.17 0.09 750 1.5 137⫻137 0.21a 0.11a 0.05a 1200 1.0 206⫻206 0.17a 0.09a 0.04a 1500 1.2 172⫻172 0.14a 0.07a 0.04a 1800 1.6 126⫻126
aIntroduction of共correctable兲 interlacing errors due to bidirectional scanning. Scanning modes indicate whether 1, 2, or 4 lines are scanned at the same time. Double and quadruple modes increase scanning speed, but decrease spatial resolution in vertical direction. FOV: field of view; image acquisition times assume frames of 256⫻256 pixels.
Fig. 1 Recording of trigger pulse, modified blood pressure, and respiration signals. A recording of the output signals from the trigger unit共modified blood pressure and respiration兲 and the trigger pulses derived from them. Data were acquired in a ventilated mouse. The blood pressure signal is modified as it is connected to an ECG-input. Nevertheless, it can be used as input for triggering.共a兲: Trigger settings and generation of trigger pulses; thresholds for respiration and modified blood pressure are indicated by horizontal dashed lines. When the respiration signal is below threshold 共expiration兲, triggering is enabled 共gray bars兲. During the first cardiac cycle a trigger pulse is generated, as triggering was enabled when modified blood pressure crossed threshold upwards共vertical dotted line兲. The next two cardiac cycles, however, did not lead to the generation of a trigger pulse, as the modified blood pressure rose above threshold when triggering was disabled. In the fourth cycle, a trigger pulse is again generated. A trigger delay could be set and is marked by asterisks. The settings for thresholds and delay were determined empirically. Motion artifacts were smallest when imaging in the diastolic phase of the cardiac cycle.共b兲: A recording for the duration of 6 s, clearly showing that trigger pulses are generated in an irregular pattern.
2.6 Triggering
The respiration sensor and one of the blood pressure sensors were connected to an electrocardiogram 共ECG兲-trigger unit 共Rapid Biomedical, Würzburg, Germany兲, originally designed for application in magnetic resonance imaging. However, an ECG could not be used as input, since positioning of an ob-jective lens on the animal strongly disturbed the recorded electrical activity. Therefore, the blood pressure signal derived from the femoral artery was fed into the ECG input, which resulted in modification of the signal. The output trigger sig-nal was coupled to the trigger/synchronizer input port of the instrumentation control unit of the TPLSM system.
The trigger unit displayed both respiration and modified blood pressure signals, and a threshold could be set for each 共Fig.1兲. The blood pressure signal was used as the primary
determinant for the trigger moment, aiming to generate one trigger pulse per cardiac cycle. Furthermore, the influence of respiration on motion artifacts was minimized using the res-piratory signal. Whenever the resres-piratory signal exceeded its threshold 共during inspiration兲, triggering was disabled. If, however, the respiration signal was below threshold and the modified blood pressure signal crossed its threshold while ris-ing, a trigger pulse was generated, initiating the acquisition of a single optical section. A trigger delay for both blood pres-sure and respiration could also be set. This simplified the search for an optimal trigger moment 共during diastole and expiration兲 without the need for adjustment of the actual blood pressure signal, respiration signal, or thresholds.
Trigger pulse, modified blood pressure, and respiration sig-nals from the trigger unit and absolute blood pressure signal were digitally recorded using the acquisition system M-PAQ in combination with the acquisition software package IDEEQ 共IDEE, Maastricht, The Netherlands兲. The sampling rate was 1000 Hz. Figure1shows such a recording for the duration of 6 s. Total frame time, start time of each subsequent image, and time gap between two subsequent optical sections 共frames兲 were recorded using the TPLSM acquisition software 共Lasersharp 6.0; Biorad兲.
2.7 Image Processing
Image reconstructions were performed using the Image-Pro Plus 6.0 software 共Media Cybernetics Inc., Silver Spring, Maryland兲. To further improve overall image quality, interlac-ing errors were corrected if needed共see results兲. Furthermore, Fourier transformation was performed to discriminate the original image frequencies共that lie around the origin, i.e., in the corners of the periodic spectrum plot兲 from higher noise and interlacing frequencies. The undesirable higher frequen-cies were then filtered out by multiplying the Fourier-spectra with a mask based on a 2-D Gaussian weighted function, thus improving SNR. Filtering in the Fourier domain and correc-tion for interlacing inaccuracy were performed using software tools developed with the Mathematica 6.0 software package with “Digital Image Processing” 共Wolfram Research Inc., Champaign, Illinois兲, and the “Front-end Vision” and “Math-visionTools” plugins 共Eindhoven University of Technology, The Netherlands兲.
3 Results
3.1 Cardiac and Respiratory Activity
During in vivo imaging, each image should be acquired well within one cardiac cycle to avoid image distortion共in-frame兲 and between-frame motion artifacts. In anesthetized mice, the duration of one cardiac cycle varied from 0.16 to 0.24 s 共4.2 to 6.1 Hz; reference values for nonanesthetized mice: 0.09 s/11.1 Hz兲. This observed cardiac depression is a known side effect of the applied ketamin/xylazin anesthesia.29 In rats, anesthetized with urethane 共which has less profound effects on heart rate兲, the cardiac cycle duration varied from 0.25 to 0.30 s 共3.3 to 4.0 Hz; reference values 0.17 s/5.9 Hz兲.30
Anesthetized animals were ventilated; rats at a rate of 120 min−1 共2.0 Hz兲 and mice at a rate of 200 to 240 min−1
共3.3 to 4.0 Hz兲 with a tidal volume of 200 to 250l. Some mice were allowed to breathe spontaneously, showing an anesthesia-induced respiratory depression over time 共mean respiration rate 161⫾35 min−1 at the start of imaging and
134⫾32 min−1at the end of the experiment兲. Therefore,
ex-perimentation time was limited in these animals. On the other hand, arterial movement due to breathing had a lower fre-quency in these mice and could thus be overcome more easily. Results obtained in artificially damaged carotid arteries were obtained this way. A drawback of unventilated triggering is a less regular respiratory activity, which might impede accurate triggering.
3.2 Nontriggered In Vivo Imaging
In view of the heart rates already given, image acquisition times should be shorter than0.16 s in mice and 0.25 s in rats to limit motion distortion. Such short image acquisition times can be achieved either by increasing the line scan rate or decreasing the number of lines per image共matrix size兲. The first method, however, decreases the pixel dwell-time at the cost of a lower SNR and, at higher rates, is accompanied by an automatic optical zoom resulting in a smaller FOV共Table
1兲. The second method results in lower pixel resolution, which
can be resolved only by using an equivalent optical zoom. Comparison of several typical acquisition rates for imaging of an ex vivo mounted artery revealed that imaging at 2 to 3 Hz yielded optical sections with a sufficient large FOV 共no optical zoom is induced兲 and relatively good overall im-age quality. However, this frequency was not high enough for
in vivo imaging since acquisition time exceeded a complete
cardiac cycle and therefore all motions within this cycle were captured in each single image 共Fig. 2 and Video 1; mouse carotid artery兲.
As published before, increasing the image acquisition rate
in vivo eliminates large motion artifacts from at least some of
the optical sections in a time series.11 Nevertheless, most of the images obtained this way are of low quality due to move-ments of the sample, loss of focus on the sample, or even complete disappearance of the vessel from the FOV.
3.3 Triggered In Vivo Imaging
Triggered image acquisition of each optical section resulted in subsequent images that共almost兲 all display the same part of the artery during the same phase of movement. Consequently,
the obtained optical sections are mutually comparable. As in untriggered imaging, higher frame rates yielded better results than rates of the order of cardiac rates, as images are acquired in a shorter part of the cardiac cycle. Therefore, a more stable period can be found using shorter image acquisition times 共Fig.3and Video 2; rat renal artery, image acquisition time 0.17 s; cardiac cycle duration 0.25 s兲.
Series of subsequent optical sections with the smallest im-pact of image distortion and between-frame motion distur-bances were obtained when images were acquired within the diastolic phase of the cardiac cycle. However, trigger pulses were generated at peaks of the modified blood pressure signal. Therefore, a trigger delay was used to start image acquisition in diastole, which could easily be introduced via the trigger unit. The amount of delay required for stable imaging was determined empirically. The timing of image acquisition in 共part of兲 the systolic phase of the cardiac cycle resulted in images with large motion artifacts and strong deformations of the tissue, even at higher imaging rates共not shown兲.
In rat renal arteries, triggered imaging often resulted in more stable images and smaller motion disturbances than in mouse carotid arteries. This finding was to be expected as the more distally located renal artery suffers less from thoracic movement or deformation due to respiration. Also, the heart rate in rats was lower.
3.4 Image Processing
The overall image quality suffered from the fast acquisition rates that affect the SNR, due to short pixel dwell-times关Fig.
4共a兲兴. The SNR was enhanced using Fourier spectrum-based
noise filtering关Fig.4共c兲兴. Additionally, fast image acquisition
using scan rates of⬎750 lines/s 共Table1兲 resulted in images
that appeared to exhibit stretched details in the horizontal di-rection. To reduce total acquisition time, odd scan lines of the matrix are automatically scanned from left to right and even scan lines from right to left 共bidirectional scanning兲, instead of scanning each horizontal line from left to right 共unidirec-tional scanning; standard for lower frame rates兲. Image analy-sis of bidirectionally scanned images revealed that in com-parison with the odd scan lines, the even scan lines of the image matrix were shifted in the horizontal direction共varying from 3 to 10 pixels兲, causing blurred images. Correction of
this interlacing inaccuracy resulted in remarkably sharper im-ages关Fig.4共b兲兴. As a consequence, the image matrix size was
also reduced in the horizontal direction, reflecting a slightly smaller FOV.
3.5 In Vivo Molecular Imaging of Exposed Subendothelial Collagen
In a subset of experiments, the carotid artery of mice was damaged to expose the thin subendothelial collagen sheet, which was labeled using CNA35-conjugated quantum dots.27 Propidium iodide共PI兲 was used as a cell viability assay. The damaging procedure dramatically increased the number of PI positive共i.e., nonviable兲 cells across the vessel wall, including the endothelium关Figs.5共a兲and5共b兲兴. Furthermore,
subendot-helial collagen, which is normally covered by endotsubendot-helial cells and can not be reached by CNA35-QD525, was now
Fig. 2 Nontriggered in vivo TPLSM at normal共ex vivo兲 scan rates. Three subsequent optical sections 共a兲 to 共c兲 of a left carotid artery of a C57Bl6/J mouse obtained in vivo without application of external triggering. Image acquisition time was 0.43 s共1200 lines/s; normal scanning mode; 512 ⫻512 pixels兲. The schematic drawing on the left indicates the position of the optical sections in the vessel wall; vSMC, vascular smooth muscle cell; FOV, field of view; bars indicate 20m. Cell nuclei共SYTO13, green兲 and extracellular matrix 共SHG of adventitial collagen, blue; autofluo-rescence of elastin, red兲 are visible. All sections are disturbed by motion artifacts, which cause the arterial wall 共blue arrow兲 to appear as a curved structure. Moreover, every optical section contains different parts of the共moving兲 vessel wall. The typical morphology and orientation of the smooth muscle cell nuclei共green兲 is hardly recognizable; the position of the lumen is unclear.
Video 1 Nontriggered in vivo TPLSM at normal共ex vivo兲 scan rates. Time series recorded in a left carotid artery of a C57Bl6/J mouse in
vivo without application of external triggering. Image acquisition time
was 0.43 s共1200 lines/s; normal scanning mode; 512⫻512 pixels兲. The FOV was approximately 200⫻200m. Cell nuclei 共SYTO13, green兲 and extracellular matrix 共SHG of adventitial collagen, blue; autofluorescence of elastin, red兲 are visible. All sections are disturbed by motion artifacts which cause the arterial wall to appear as a curved structure. Moreover, every optical section contains different parts of the moving vessel wall. The typical morphology and orientation of the smooth muscle cell nuclei共green兲 is hardly recognizable; the position of the lumen is unclear 共QuickTime, 817 KB兲. 共Color online only.兲
exposed and could be labeled with CNA35-QD525 resulting in a bright green labeled layer. This thin layer was visualized at a subcellular level in vivo, and is clearly located between the smooth muscle and endothelial cell nuclei 关Fig. 5共c兲兴,
which can be distinguished based on morphology and location in the vessel wall.1The collagen in the adventitial layer 共as shown by SHG兲 was not labeled with CNA35-QD525, which indicates that the elastic laminae were still intact and pre-vented CNA35-QD525 labeling over the vessel wall.
4 Discussion
We showed the feasibility of in vivo imaging of structures in the wall of large elastic arteries of mice and rats at a subcel-lular resolution. Moreover, we visualized the thin collagen sheet present under the endothelium after damaging the vessel
Fig. 3 Triggered in vivo TPLSM at high scan rates. Five successive
optical sections共a兲 to 共e兲 of the left renal artery of a Wistar-Kyoto rat in
vivo obtained by triggered acquisition at an image acquisition time of
0.17 s共1500 lines/s; normal scanning mode; 256⫻256 pixels兲. Car-diac cycle duration was 0.25 s. Schematic drawing displays the posi-tion of the optical secposi-tions; vSMC, vascular smooth muscle cell; FOV, field of view; bars indicate 20m. Artery was labeled topically for cell nuclei共SYTO13, green兲 and systemically for cytoplasm of 共blood兲 cells and platelets共acridin red, orange兲. The successive optical sec-tions contain the same part of the artery. In the tunica adventitia, collagen共SHG, blue兲 and nuclei 共arrowhead兲 are visible; vSMC nuclei 共white arrow兲 are observable in the tunica media. Moreover, an ad-hesive blood cell共encircled by white dotted line兲 is visible against the vessel wall in several共⬎10兲 successive images. The lumen appears orange due to circulating unbound acridin red.
Fig. 4 Image enhancement. The effects of image processing applied to one of the optical sections of the series of images as shown in Fig.3;共a兲 raw image of the data file, as shown in fig.3, the image appears fuzzy and the smooth muscle cell nuclei共white arrow兲 are hardly recognizable as separate structures;共b兲 the same optical section after correction for inaccurate line positioning 共interlacing error兲, the image already appears sharper and the smooth muscle cell nuclei共white arrow兲 appear separated and an adhesive blood cell 共encircled by white dotted line兲 is more clearly visible; and共c兲 filtering in the Fourier domain further improves SNR of the image. Also, the nuclei of smooth muscle cells 共white arrow兲 are more apparent.
Video 2 Triggered in vivo TPLSM at high scan rates. Stable recording of the left renal artery of a Wistar-Kyoto rat in vivo obtained by trig-gered acquisition at an image acquisition time of 0.17 s. The FOV was approximately 200⫻200m. The artery was labeled topically for cell nuclei 共SYTO13, green兲 and systemically for cytoplasm of 共blood兲 cells and platelets 共acridin red, orange兲. The nuclei of the smooth muscle cells are labeled by both probes共yellow兲. SHG of collagen 共blue兲 is visible in the tunica adventitia. Images show no distortion and between-frame motion is very small due to triggered acquisition. 共QuickTime, 709 KB兲. 共Color online only.兲
关URL: http://dx.doi.org/10.1117/1.3281672.2兴. Megens et al.: In vivo high-resolution structural imaging of large arteries in small rodents…
wall. It is the first time the subendothelial collagen layer has been imaged in vivo in vessels of this size. Application of fluorescently labeled CNA35 combined with in vivo triggered TPLSM imaging might prove a valuable tool to investigate the role of subendothelial collagen in the onset or progression of共unstable兲 atherosclerotic plaques.12,28Furthermore, it dem-onstrates the enormous potential for stable detection of small vascular structures deep in the tissue, such as subcellular ath-erosclerotic plaque components,28 and the endothelial glycocalyx.31
In vivo imaging in larger arteries is severely challenged
due to in-frame and between-frame motion artifacts. We showed that a combination of short image acquisition times and triggering on cardiac and respiratory cycles can for the most part overcome these problems 共cf., Videos 1 and 2兲.
Although image distortion may still occur共Fig.5兲, the
influ-ence of between-frame vessel wall movement on image sta-bility is greatly reduced.
Triggering on blood pressure signal proved to be most ef-fective to improve image quality as vessel motion is mainly governed by pulse wave deformation共cardiac activity兲. This is especially true for rat renal arteries. Therefore, application of a more sophisticated solid state pressure transducer catheter 共Millar transducer without fluid column, as applied in Ref.
32兲, which yields more stable and accurate blood pressure
signals, may enhance the precision of triggering and, thus, provide more accurate timing of image acquisition. This would reduce between-frame motion artifacts even further.
Image distortion due to vessel motion was reduced by ap-plying shorter image acquisition times共typically 0.17 s in rat renal arteries and0.11 s in mouse carotid arteries兲. Assuming that the duration of the diastolic phase is about half the car-diac cycle, stable images can be acquired in animals with heart rates varying from 2.9 to 4.5 Hz 共0.34 and 0.22 s per cardiac cycle, respectively兲 with these settings. If necessary, image acquisition times could be shortened to match higher heart rates 共Table 1兲. However, there is a trade-off between
reduction of distortion and SNR.
Short image acquisition times also dictate small image ma-trix sizes. As a consequence, pixel resolution is not optimal. Furthermore, high excitation powers are required to generate sufficient signal. This will lead to increased photodegradation of the fluorescent markers, which causes the SNR to drop over time. In the data presented here, however, photodegrada-tion was not observed to hamper experiments significantly. Interestingly, quantum dots are hardly vulnerable to photobleaching,33 making them ideal candidates for more lengthy in vivo measurements.
The experiments were performed using a combination of different fluorescent markers, each with their own excitation maximum. At the chosen excitation wavelengths 共800 to 840 nm兲, this meant that the probes could all be ex-cited sufficiently but not optimally. Again, quantum dots are the exception to this rule, as they have broad excitation spec-tra, with only very small emission peaks.
Theoretically, the axial resolution of our system would be around0.9m, depending on the excitation wavelength and the NA of the objective used. As discussed elsewhere,1,28 a more realistic value would be 1.5 to 2.0m, depending on the location in the vessel. This can for the larger part be ex-plained by a mismatch between the refractive index of the vessel wall components and the objective, which accumulates with increasing depth. Furthermore, the axial resolution will deteriorate when imaging deeper in共scattering兲 tissue, albeit to a lesser extent than with conventional microscopy.
Using double or quadruple scanning modes to reduce im-age acquisition time limited spatial resolution in the vertical direction 共y direction兲. Still, even nonoptimal by aligned structures could be imaged at a sufficient resolution共Fig.5兲.
Applying short image acquisition times also decreased the SNR. Using simple and straightforward image processing
al-Fig. 5 Imaging of subendothelial collagen prior to and after
mechani-cal damage. Optimechani-cal sections of a left carotid artery of a C57Bl6/J mouse obtained in vivo with application of external triggering. Images were recorded at double scanning mode, resulting decreased spatial resolution in the vertical direction 共y direction兲. Image acquisition time was 0.11 s 共1200 lines/s; double scanning mode; 256 ⫻256 pixels兲. Cardiac cycle duration was 0.45 s. Schematic drawings on the left indicate the position of the optical sections in the vessel wall; PI, propidium iodide; FOV, field of view; ROI, region of interest; bars indicate 25m. Mouse carotid artery in vivo after injection of PI 共red兲 and CNA35-QD525 共green兲 共a兲 prior to and 共b兲 after pinching. 共a兲 Prepinching; some PI-positive cells 共red兲 can be found in the ad-ventitia共blue SHG, arrowhead兲 due to surgery; 共b兲 postpinching; the image distortion共white arrow兲 in the top part indicates that part of the image was not recorded during the diastolic phase; by selecting an ROI共blue dotted area兲 a stable window can be defined; and 共c兲 ROI from共b兲. Almost all cell nuclei are PI-positive 共red; i.e., have a com-promised outer cell membrane兲 and, therefore, a labeled collagen sheet共green兲 can be observed located between endothelial cell nuclei 共round and closest to the lumen; blue arrows兲 and smooth muscle cell nuclei共elongated; purple arrow兲. Adventitial collagen is not labeled by quantum dots, but can be identified by SHG共blue, arrowhead兲.
gorithms, the SNR could be improved. Other image process-ing techniques could be explored to guarantee optimal post-acquisition SNR improvement. Acquisition SNR can also be improved by narrowing the pulse width of the excitation light, which would enhance the probability of the two-photon excitation.34As a consequence, signal strength and, thus, SNR increases, which is beneficial for the image quality of accel-erated image acquisition. On the other hand, a smaller pulse width would result in increased photodegradation of the fluo-rescent marker, thus counteracting the increase in signal strength.
Novel high-speed two-photon microscopes22,35enable im-age acquisition at video rates and will therefore be less ham-pered by image distortion while providing good SNRs. Still, triggered acquisition is required to limit between-frame mo-tion artifacts. The present triggering method can easily be applied to these systems.
As already discussed, setting up in vivo experiments based on two-photon microscopy means balancing acquisition speed with heart rate and SNR, excitation wavelength with excita-tion spectra of fluorescent probes and penetraexcita-tion depth, and excitation power with penetration depth and photodegradation 共length of experiment兲. The limitation of the presented system is sampling rate, since the generation of a trigger pulse is dependent on both the cardiac and the respiratory cycle. This means that the delay between images may vary between one and three to four cardiac cycles共Fig.1兲. All taken together,
the actual sampling rate of triggered image acquisition in the present system is⬃1 to 4 Hz. This is relatively low for im-aging of fast functional processes in the vessel wall, such as calcium or nitric oxide dynamics or tracking of moving ob-jects such as blood cells. Nevertheless, slow or stable interac-tions can still be studied, as shown in Fig. 3. This is also underlined inVideo 3labeled topically and systemically with SYTO13. The video clearly shows cells with lobed nuclei, acquired from a carotid artery of a ventilated mouse. Cell nuclei were slowly moving in the lumen near the vessel wall. These optical sections were obtained at the end of the experi-ment, where the blood pressure of the mouse had dropped below 50 mmHg, while heart rate was unaltered. Although the physiological relevance of these images should be
inter-preted with care, they do underline the vast potential of trig-gered in vivo TPLSM imaging.
In conclusion, the presented method creates new opportu-nities for in vivo共molecular兲 imaging at a subcellular level of structures in the共diseased or damaged兲 arterial wall. Further-more, 3-D imaging of the arterial wall might be possible, especially when the stability and accuracy of triggering on blood pressure is optimized. In addition, it can be used as a platform for in vivo testing and validation of novel molecular agents共e.g., targeted quantum dots33兲 as it provides detailed information regarding the behavior of such agents in large arteries in vivo. We showed this tool to be very suitable for CNA35-mediated in vivo collagen imaging.
Acknowledgments
We thank Evelien Hermeling and Jeroen Hameleers from Maastricht University, Department of Biomedical Engineer-ing, for their help with signal analysis and technical assis-tance.
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Video 3 Triggered in vivo TPLSM provides subcellular resolution. Time series recorded in a carotid artery of a ventilated mouse. Cell nuclei were labeled topically and systemically with SYTO13共green兲. Cells with lobed nuclei can be distinguished from smooth muscle cells 共oriented in vertical direction兲, and are slowly moving in the lumen near the vessel wall. These optical sections were obtained at the end of the experiment, where the blood pressure of the mouse had dropped below 50 mmHg, while heart rate was unaltered. The physi-ological relevance of the visualized phenomenon is still unclear, but it shows the subcellular resolution of triggered TPLSM in vivo. 共Quick-Time, 1.5 Mb兲. 共Color online only.兲
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